CN113683717A

CN113683717A - Micron-sized aggregation-induced emission polymer microsphere and preparation method and application thereof

Info

Publication number: CN113683717A
Application number: CN202111194628.6A
Authority: CN
Inventors: 唐本忠; 胡亚新; 许跃; 王志明; 刘勇; 谢莉
Original assignee: Institute Of Cluster Induced Luminescence South China University Of Technology Dawan District Guangdong Province
Current assignee: Institute Of Cluster Induced Luminescence South China University Of Technology Dawan District Guangdong Province
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2021-11-23
Anticipated expiration: 2041-10-13
Also published as: CN113683717B

Abstract

The invention discloses a micron-sized aggregation-induced emission polymer microsphere and a preparation method and application thereof; the preparation method comprises the following steps: after the stabilizer solution and the monomer solution are mutually dissolved, heating and stirring, centrifugally purifying and diluting to obtain a seed microsphere solution; adding a swelling agent, performing ultrasonic treatment, and performing swelling treatment to obtain an activated swelling solution; adding a hydrophobic monomer, a functional monomer, an AIE molecule and an initiator into a surfactant aqueous solution, and carrying out ultrasonic treatment to obtain a monomer emulsified solution; adding the monomer emulsion solution into the activated swelling solution, swelling, adding a stabilizer and a water-phase polymerization inhibitor, reacting, and centrifugally purifying to obtain the micron-sized aggregation-induced emission polymer microspheres. The invention realizes the preparation of seed microspheres with high conversion rate and narrow particle size distribution by optimizing the formula and operation process of dispersion polymerization and seed swelling polymerization; the prepared polystyrene microsphere has the advantages of complete microsphere appearance, good monodispersity, narrow particle size distribution and more than 10 mu m.

Description

Micron-sized aggregation-induced emission polymer microsphere and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a micron-sized aggregation-induced emission polymer microsphere, a preparation method thereof and application thereof in preparation of coupled biomolecules for fluorescence tracking.

Background

The micron-sized fluorescent microspheres have wide application in some high and new technical fields such as immunoassay, biochemistry, analytical chemistry, carrier catalysis, information, microelectronics, liquid crystal television and the like due to uniform physicochemical properties. The polystyrene fluorescent microspheres with the particle size of 1-20 microns are widely applied to the fields of cell separation, biological modeling, coupling biomolecule detection of cytokines, gene sequences and the like. However, the conventional fluorescent molecules have a fluorescence aggregation induced quenching (ACQ) phenomenon at high concentration, so that the application is limited to a certain extent. In 2001, the team of the Thanksonics proposed a fluorescent molecule with aggregation-induced emission (AIE), which, unlike ACQ molecules, fluoresces weakly in good solvents and strongly fluoresces due to the restricted rotation within the molecule when aggregated in poor solvents, and AIE polymer materials brought a new application scheme for biological modeling and fluorescence tracking.

At present, various methods have been reported to prepare micron-sized polystyrene fluorescent microspheres, for example, a zhuan subject group [ cn104262811a ] prepares blank microspheres by soap-free emulsion polymerization, and then prepares fluorescent microspheres by using a swelling method to load organic dyes, and a zhuan subject group [ j. The method has the advantages of wide application range, no requirement on dye structure, complex operation of the swelling adsorption method, low efficiency and great manual interference on batch stability.

At present, a method for synthesizing micron-sized AIE polystyrene fluorescent microspheres by a stable process is provided, and based on the problems, a simple and convenient method is provided for preparing the micron-sized AIE polystyrene fluorescent microspheres.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a process for stably preparing the polystyrene AIE fluorescent microspheres with the particle size of more than 10 microns, and the prepared polystyrene AIE fluorescent microspheres have the characteristics of high fluorescence intensity, narrow particle size distribution, high sphericity and the like.

The invention uses a two-step seed swelling method to prepare fluorescent microspheres, firstly uses dispersion polymerization to prepare polystyrene seed microspheres, and then adds a monomer solution for dissolving AIE molecules to carry out seed swelling polymerization, thereby preparing micron-sized AIE polystyrene fluorescent microspheres with high fluorescence intensity, narrow particle size distribution and high sphericity.

The technical scheme adopted by the invention is as follows:

a preparation method of micron-sized aggregation-induced emission polymer microspheres comprises the following steps:

(1) dissolving a stabilizer in a solvent to obtain a stabilizer solution;

(2) mutually dissolving a hydrophobic monomer and an initiator to obtain a monomer solution;

(3) mutually dissolving the stabilizer solution in the step (1) and the monomer solution in the step (2), heating and stirring, centrifugally purifying, and diluting to obtain a seed microsphere solution;

(4) adding a swelling agent into the seed microsphere solution obtained in the step (3), and performing ultrasonic treatment and swelling treatment to obtain an activated swelling solution;

(5) adding a surfactant into water to dissolve to obtain a surfactant aqueous solution, adding a hydrophobic monomer, a functional monomer, an AIE molecule and an initiator into the surfactant aqueous solution, and performing ultrasonic treatment to obtain a monomer emulsified solution;

(6) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), and swelling to obtain a monomer swelling solution; adding a stabilizer and a water-phase polymerization inhibitor, reacting, centrifuging and purifying to obtain the micron-sized aggregation-induced emission polymer microspheres.

Preferably, the stabilizer solution in the step (1) is preheated at 50-80 ℃ for 10-40 min;

preferably, the mass of the stabilizer in the step (1) is 0.1-10% of that of the solvent;

preferably, the stabilizer in step (1) is selected from one of the following: polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, dextrin, polyethylene glycol;

preferably, the solvent in step (1) is selected from one of methanol, ethanol, propanol, butanol, pentanol or a mixed solution of ethanol and water.

Preferably, the hydrophobic monomer in the step (2) accounts for 5-40% of the mass of the solvent in the step (1), and the initiator accounts for 0.1-10% of the mass of the solvent in the step (1);

preferably, the hydrophobic monomer in step (2) is selected from one of the following: styrene, acrylic acid, methyl acrylate, methyl methacrylate, methacrylic acid;

preferably, the initiator in step (2) is selected from one of the following: azobisisobutyronitrile, dibenzoyl peroxide, azobisisovaleronitrile, cumene hydroperoxide, azobisisoheptonitrile, tert-butyl peroxypivalate, diisopropyl peroxydicarbonate, and dilauroyl peroxide.

Preferably, the heating and stirring temperature in the step (3) is 40-90 ℃, and the time is 16-36 h;

preferably, the speed of the centrifugal purification in the step (3) is 1000 rpm-5000 rpm, and the time of the centrifugal purification is 3 min-40 min;

preferably, the mass fraction of the seed microsphere solution in the step (3) is 0.5-1.5%; further preferably, the mass fraction of the seed microsphere solution in the step (3) is 1%.

Preferably, the mass of the swelling agent in the step (4) accounts for 0.1-5% of the mass of the seed microsphere solution;

preferably, the power of the ultrasonic treatment in the step (4) is 25W-950W, the time is 0.5 min-60 min, and the temperature is 0 ℃ to 5 ℃;

preferably, the temperature of the swelling treatment in the step (4) is 20-60 ℃, and the time is 6-24 h;

preferably, the temperature rise rate of the swelling treatment in the step (4) is 0.5-5 ℃/min;

preferably, the swelling agent in step (4) is selected from one of the following: dibutyl phthalate, trichloromethane, dichloromethane, toluene, tetrahydrofuran, acetone and cyclohexane.

Preferably, the power of the ultrasonic treatment in the step (5) is 25W-950W, and the time is 0.5 min-60 min; the temperature of ultrasonic treatment is 0-5 ℃;

preferably, the mass of the surfactant in the step (5) is 0.1-10% of the mass of water, the mass of the functional monomer is 0.1-50% of the mass of the hydrophobic monomer, the mass of the AIE molecule is 0.5-50% of the mass of the hydrophobic monomer, the mass of the initiator is 0.1-10% of the mass of the hydrophobic monomer, and the mass of the hydrophobic monomer is 2-30% of the mass of water.

Preferably, the surfactant in step (5) is selected from one of the following: sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, dodecyl amino propionic acid, octadecyl dihydroxyethyl amine oxide, and tween-20;

preferably, the hydrophobic monomer of step (5) is selected from one of the following: styrene, methyl acrylate, methyl methacrylate;

preferably, the functional monomer in step (5) is selected from one of the following: acrylic acid, methacrylic acid, hydroxyethyl methacrylate, itaconic acid;

preferably, the AIE molecule of step (5) is selected from at least one of the following AIE-1 to AIE-18 molecules:

preferably, the initiator in step (5) is selected from one of the following: azobisisobutyronitrile, dibenzoyl peroxide, azobisisovaleronitrile, cumene hydroperoxide, azobisisoheptonitrile, tert-butyl peroxypivalate, diisopropyl peroxydicarbonate, and dilauroyl peroxide.

Preferably, the swelling temperature in the step (6) is 20-60 ℃, and the swelling time is 3-10 h;

preferably, the temperature rise rate of the swelling in the step (6) is 0.5-5 ℃/min;

preferably, the reaction temperature in the step (6) is 40-100 ℃, and the reaction time is 10-36 h;

preferably, the temperature rise rate of the reaction in the step (6) is 0.5-5 ℃/min;

preferably, the stabilizer in step (6) is selected from one of the following: polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol;

preferably, the aqueous phase polymerization inhibitor in the step (6) is selected from one of the following: cuprous chloride and sodium nitrite.

The micron-sized aggregation-induced emission polymer microsphere prepared by the preparation method.

The micron-sized aggregation-induced emission polymer microsphere is applied to the preparation of coupled biomolecules for fluorescence tracking.

According to the invention, through research, in the dispersion polymerization process, the stirring speed influences the particle size and the particle size distribution of the microspheres, the unreacted monomers have certain viscosity, the microspheres are easy to adhere due to collision, the steric hindrance benefit of the stabilizer is needed to prevent the microspheres from agglomerating, and the stable effect is damaged due to the overhigh stirring speed, so that the particle size and the particle size distribution of the microspheres are enlarged. Therefore, the stirring rate is selected depending on the scale of the reaction system and the range of application. Meanwhile, the selection of the solvent plays an important role in the particle size of the microsphere, and as the polarity of the solvent increases, the shorter the soluble critical long chain is, the less time is required for precipitation from the solvent, the faster nucleation is, the more primary nuclei are obtained, and the smaller the particle size of the formed microsphere is, so that the proper polarity of the solvent needs to be selected according to the requirement of the particle size.

In the step (1), in order to ensure the uniformity of the particle size of the microspheres, the stabilizer is preferably polyvinylpyrrolidone, and the solvent is preferably a mixed solution of ethanol, ethanol and water;

in the step (3), in order to maintain the steric hindrance effect of the stabilizer from being damaged, the stirring speed of the polymerization reaction is preferably 20rpm to 200rpm, and is more preferably 100 rpm;

in the step (3), because the particle size of the prepared microspheres is micron-sized, and secondary nucleated nano-scale spheres exist, the rate is not high during centrifugal purification, and the centrifugal rate is preferably 1000rpm to 5000 rpm; the centrifugal speed is more preferably 3000rpm, and the centrifugal time is preferably 3 min-40 min; the centrifugation time is more preferably 10 min.

According to the invention, through research, in the method for preparing the micron-sized fluorescent microspheres by using the seed swelling method, the type and the amount of the swelling agent have great influence on the stability of a reaction system, the size of the microspheres, the particle size distribution and the conversion rate. In general, a polymer microsphere formed by hard monomers is difficult to directly absorb a large amount of monomers and polymerize into a microsphere with a large particle size, and the monomer absorption capacity of the microsphere swelled by a proper amount of a low water-soluble compound is greatly improved, so that the swelling effect of the microsphere is greatly influenced by selecting a proper swelling agent in the seed swelling process. Meanwhile, as the amount of the swelling agent is increased within a certain range, the stability of the reaction system is better, the particle size distribution of the final microspheres is narrower, but the excessive swelling agent can cause the surface charge layer of the microspheres to be damaged, so that the stability of the reaction system is poor, the particle size of the final microspheres is increased, and the particle size distribution is widened. Therefore, the proper amount of swelling agent in the system needs to be determined according to the required particle size and distribution of the microspheres.

In step (4), the swelling agent is preferably one of the following: dibutyl phthalate, trichloromethane and toluene.

In the steps (4) and (5), because the swelling agent and the monomer have certain volatility, the reaction temperature needs to be controlled in the ultrasonic emulsification process, generally, the crude emulsion is placed in an ice bath in the ultrasonic process, the ultrasonic power is preferably 50W-600W, and the ultrasonic time is preferably 5 min-30 min.

In step (5), the micron-sized fluorescent microspheres are mainly used for in vitro applications such as biological modeling and cell separation, and the hydrophobic monomer should preferably be one of the following: styrene and methyl methacrylate, and styrene is more preferable.

In the step (5), in view of providing sufficient reaction sites for subsequent modification and ensuring that the particles have better colloidal stability, the amount of the carboxyl functional monomer is preferably 1 to 10 percent of the mass amount of the hydrophobic monomer.

In step (6), the aqueous phase polymerization inhibitor is preferably sodium nitrite in order to minimize the influence of the aqueous phase polymerization inhibitor on the colloidal stability.

According to the invention, through research, the temperature can influence the thermodynamic motion of each particle in the swelling polymerization process, and with the rise of the temperature, the swelling agent is favorably diffused into the seed microspheres, and the seed microspheres are favorably used for absorbing monomers; on the other hand, too high temperature is not favorable for the dispersion of the swelling agent and the monomer in the water phase, and the swelling agent and the monomer are easy to be aggregated into small droplets, so that the swelling effect of the microspheres is seriously influenced, and even the bonding deformation of the polymer is caused. Therefore, the shape of the microsphere can be ensured to be intact by selecting a proper swelling temperature, and the microsphere with larger particle size can be obtained.

Researches show that the temperature rise rate has an influence on the particle size distribution of the final microspheres, the seed microspheres take a certain time to swell by the monomers, and the uneven microspheres are generated when the seed microspheres reach a higher temperature before the monomers are completely absorbed by the seed microspheres. In general, the temperature increase rate is preferably 0.5 ℃/min to 5 ℃/min, as the particle size distribution of the final microspheres is broader.

Compared with the prior art, the invention provides a novel method for preparing the polystyrene fluorescent microspheres with the particle size of more than 10 microns, the polystyrene microspheres with the particle size of 3-5 microns are prepared by dispersion polymerization, AIE molecules, an initiator, a hydrophobic monomer and a functional monomer are added, and high-brightness AIE polystyrene fluorescent microspheres with the particle size of more than 10 microns are formed by seed swelling polymerization.

The invention has the advantages that:

(1) the preparation of the seed microspheres with high conversion rate and narrow particle size distribution is realized by improving the formula and the operation process of dispersion polymerization and seed swelling polymerization;

(2) the swelling polymerization process is optimized to prepare the polystyrene microspheres with the particle size of more than 10 microns, the generation of secondary nucleation is greatly reduced by the aqueous phase polymerization inhibitor, the prepared microspheres have complete appearance, good monodispersity, narrow particle size distribution and strong fluorescence signals, and the particle size can be adjusted by adjusting the formula parameters;

(3) the micron-sized AIE fluorescent microspheres are prepared in two steps by adding AIE molecules in a swelling polymerization stage, and the emission wavelength and the emission intensity of the fluorescent microspheres can be adjusted through the load capacity and the type of the AIE fluorescent molecules;

(4) the type and the dosage of the functional monomer are simply changed, and the surface of the microsphere is modified with different functional groups, including: amino, carboxyl and hydroxyl, and is applied to different biological fields.

Drawings

FIG. 1 is a distribution diagram of the particle size of seed microspheres prepared in example 1.

FIG. 2 is a scanning electron micrograph of the seed microspheres prepared in example 1.

FIG. 3 shows the particle size distribution of the AIE polystyrene fluorescent microspheres prepared in example 1.

FIG. 4 is a scanning electron micrograph of the AIE polystyrene fluorescent microspheres prepared in example 1.

FIG. 5 is a spectrum of AIE polystyrene fluorescent microspheres prepared in example 1.

FIG. 6 is a scanning electron microscope image of the seed microspheres prepared in comparative example 1.

FIG. 7 is a scanning electron micrograph of the seed microspheres prepared in comparative example 2.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

The micron-sized AIE polystyrene fluorescent microspheres are used for feasibility research of fluorescence coupling diatom in drowning diagnosis, the AIE fluorescent microspheres are adsorbed in diatom shell lines to prepare diatom drowning liquid with AIE fluorescence effect, rats are drowned in the drowning liquid, and the diatom with AIE fluorescence effect enters organs such as lungs, livers and kidneys of the rats along with the drowning liquid. Taking lung, liver and kidney, preparing into slices, observing the distribution of diatom contained in each organ under a fluorescence microscope, and testing a fluorescence signal value by using a self-contained detector.

Example 1

(1) Weighing 0.8g of polyvinylpyrrolidone stabilizer, dissolving in 50g of ethanol to obtain a stabilizer solution, adding the stabilizer solution into a 250mL four-neck flask, and stirring and preheating at the rotating speed of 50rpm at 70 ℃ for 20 min;

(2) weighing 0.2g of azobisisobutyronitrile initiator, adding the initiator into 10g of styrene monomer, stirring and mixing at the rotating speed of 400rpm at 40 ℃, and uniformly mixing to obtain a monomer solution;

(3) adding the monomer solution into the stabilizer solution, introducing nitrogen to carry out sweeping for 15min, and stirring and reacting for 24h in an oil bath kettle at 70 ℃ at the rotating speed of 50 rpm; after the reaction is finished, centrifuging for three times at the rotating speed of 5000rpm, washing for 10min each time by deionized water for multiple times to obtain a polystyrene seed microsphere solution, determining the solid content and the conversion rate, and diluting the polystyrene seed microsphere solution to the concentration of 1%.

The morphology of the polystyrene seed microspheres is observed by a scanning electron microscope, the polystyrene seed microspheres are observed to be spherical particles (as shown in figure 2), the particle size is 2.8 mu m (as shown in figure 1), the conversion rate of the seed microspheres is measured by a gravimetric method, and the conversion rate of the seed microspheres is calculated to be 92%.

(4) Weighing 0.2g of dibutyl phthalate, adding the dibutyl phthalate into 10g of seed microsphere solution, placing the mixture into an ice water bath, carrying out ultrasonic treatment for 9min at the power of 400W to obtain swelling agent solution, adding the swelling agent solution into a four-neck flask, and swelling the mixture at the temperature of 30 ℃ for 10h at the heating rate of 1 ℃/min and the rotating speed of 200rpm to obtain activated swelling solution;

(5) weighing 0.08g of sodium dodecyl sulfate, adding the sodium dodecyl sulfate into 80mL of ultrapure water to obtain an aqueous phase solution, weighing 0.2g of azodiisobutyronitrile initiator, 0.1g of methacrylic acid and 0.1g of AIE-1 molecules, adding the mixture into 10g of styrene monomer, uniformly mixing, adding the aqueous phase solution, stirring at the rotating speed of 700rpm at 40 ℃ for 15min for pre-emulsification, placing a container filled with a coarse emulsion in an ice-water bath, and performing ultrasonic treatment at the power of 400W for 9min to obtain a monomer emulsified solution;

(6) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), introducing nitrogen, cleaning for 15min, swelling for 6h at 30 ℃, heating up at a rate of 1 ℃/min, then adding 2g of polyvinylpyrrolidone stabilizer and 0.05g of sodium nitrite aqueous phase polymerization inhibitor, heating up to 80 ℃, heating up at a rate of 1 ℃/min, reacting for 24h at a rotating speed of 200rpm, centrifuging for three times at a rotating speed of 3000rpm after the reaction is finished, each time for 10min, and washing with deionized water for multiple times to obtain the micron-sized AIE polystyrene fluorescent microsphere solution.

The morphology of the AIE polystyrene fluorescent microspheres is observed through a scanning electron microscope, and the AIE polystyrene fluorescent microspheres are observed to be spherical particles (shown in figure 4), and the particle size of the AIE polystyrene fluorescent microspheres is 10-11 microns (shown in figure 3). The fluorescence spectrum of the AIE polystyrene fluorescent microsphere shows (as shown in figure 5), the emission wavelength of the AIE polystyrene fluorescent microsphere is 564nm, and the AIE polystyrene fluorescent microsphere is green fluorescence under the irradiation of an ultraviolet lamp. The fluorescent diatom screening test is used for researching the feasibility of the fluorescence-coupled diatom in drowning diagnosis, the distribution of AIE fluorescent diatom can be clearly and brightly observed under a fluorescent microscope, and the fluorescent signal value is 21309.

Comparative example 1

Using the same formulation and preparation conditions as in example 1, a polystyrene seed microsphere solution was prepared.

(4) Weighing 0.2g of dichloromethane, adding the dichloromethane into 10g of seed microsphere solution, placing the solution in an ice water bath, carrying out ultrasonic treatment for 9min at the power of 400W to obtain a swelling agent solution, adding the swelling agent solution into a four-neck flask, swelling for 10h at the temperature of 30 ℃, and obtaining an activated swelling solution at the temperature rise rate of 1 ℃/min and the rotation speed of 200 rpm;

(6) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), introducing nitrogen, cleaning for 15min, swelling for 6h at 30 ℃, increasing the temperature at a rate of 1 ℃/min, then adding 2g of polyvinylpyrrolidone stabilizer and 0.05g of sodium nitrite aqueous phase polymerization inhibitor, increasing the reaction temperature to 80 ℃, increasing the temperature at a rate of 1 ℃/min, reacting for 24h at a rotating speed of 200rpm, centrifuging for three times at a rotating speed of 3000rpm after the reaction is finished, each time for 10min, and washing with deionized water for multiple times to obtain the AIE polystyrene fluorescent microsphere solution.

As the swelling agent is a hydrophobic dichloromethane solvent, the surface layer structure of the polystyrene seed microsphere can be damaged in the swelling process, and the morphology of the AIE polystyrene fluorescent microsphere is observed by a scanning electron microscope, so that the AIE polystyrene fluorescent microsphere can be observed to be irregular in morphology, poor in particle size distribution and failed to prepare (as shown in figure 6). The fluorescent diatom screening test strip is used for researching the feasibility of fluorescent coupled diatom in drowning diagnosis, the distribution of AIE fluorescent diatom cannot be seen under a fluorescent microscope, and the fluorescent signal value is 1028.

Comparative example 2

(4) Weighing 0.2g of dibutyl phthalate, adding the dibutyl phthalate into 10g of seed microsphere solution, placing the mixture into an ice water bath, carrying out ultrasonic treatment for 9min at the power of 400W to obtain swelling agent solution, adding the swelling agent solution into a four-neck flask, swelling for 10h at the temperature of 60 ℃, and obtaining activated swelling solution at the temperature rise rate of 1.5 ℃/min and the rotation speed of 200 rpm;

(6) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), introducing nitrogen, cleaning for 15min, swelling for 6h at 60 ℃, heating up at a rate of 1.5 ℃/min, then adding 2g of polyvinylpyrrolidone stabilizer and 0.05g of sodium nitrite aqueous phase polymerization inhibitor, heating up to 80 ℃, heating up at a rate of 1.5 ℃/min, reacting for 24h at a rotating speed of 200rpm, centrifuging for three times at a rotating speed of 3000rpm after the reaction is finished, each time for 10min, and washing with deionized water for multiple times to obtain the AIE polystyrene fluorescent microsphere solution.

In the swelling process of the seed microsphere, the temperature of 60 ℃ is not favorable for the dispersion of the swelling agent and the monomer in the water phase, the swelling agent and the monomer are easy to be aggregated into small droplets, the swelling effect of the microsphere is seriously influenced, and the AIE polystyrene fluorescent microsphere is irregular in appearance, poor in particle size distribution and failed to prepare by observing the appearance of the AIE polystyrene fluorescent microsphere through a scanning electron microscope. (see fig. 7). The fluorescent diatom screening test strip is used for researching the feasibility of fluorescent coupled diatom in drowning diagnosis, the distribution of AIE fluorescent diatom cannot be seen under a fluorescent microscope, and the fluorescent signal value is 1028.

Example 2

(1) Weighing 0.05g of polyvinyl alcohol stabilizer, dissolving the polyvinyl alcohol stabilizer in a mixed solution of 40g of ethanol and 10g of water to obtain a stabilizer solution, adding the stabilizer solution into a 250mL four-neck flask, and stirring and preheating at 50 ℃ at the rotating speed of 20rpm for 10 min;

(2) weighing 0.05g of cumene hydroperoxide initiator, adding the cumene hydroperoxide initiator into 2.5g of methyl methacrylate monomer, stirring and mixing at the rotating speed of 400rpm at 40 ℃, and uniformly mixing to obtain monomer solution;

(3) adding the monomer solution into the stabilizer solution, introducing nitrogen for cleaning for 5min, and stirring and reacting for 16h in an oil bath kettle at 40 ℃ at the rotating speed of 50 rpm; after the reaction is finished, centrifuging for three times at the rotating speed of 3000rpm, washing for 10min each time by deionized water for multiple times to obtain a polymethyl methacrylate seed microsphere solution, determining the solid content and the conversion rate, and diluting the polystyrene seed microsphere solution to the concentration of 1%.

The morphology of the polystyrene seed microspheres is observed by a scanning electron microscope, the polystyrene seed microspheres are observed to be spherical particles with the particle size of 2.4 mu m, the conversion rate of the seed microspheres is measured by a gravimetric method, and the conversion rate of the seed microspheres is calculated to be 94%.

(4) Weighing 0.02g of dibutyl phthalate, adding the dibutyl phthalate into 10g of seed microsphere solution, placing the mixture into an ice water bath, carrying out ultrasonic treatment for 20min at the power of 50W to obtain swelling agent solution, adding the swelling agent solution into a four-neck flask, swelling for 6h at the temperature of 25 ℃, and obtaining activated swelling solution at the temperature rise rate of 1.5 ℃/min and the rotation speed of 200 rpm;

(5) weighing 0.1g of sodium dodecyl sulfate, adding the sodium dodecyl sulfate into 80mL of ultrapure water to obtain an aqueous phase solution, weighing 0.01g of azobisisobutyronitrile initiator, 0.2g of methacrylic acid and 0.15g of AIE-2 molecules, adding the mixture into 10g of styrene monomer, uniformly mixing, adding the aqueous phase solution, stirring at the rotating speed of 700rpm at 40 ℃ for 15min for pre-emulsification, placing a container filled with a coarse emulsion in an ice water bath, and performing ultrasonic treatment at the power of 50W for 20min to obtain a monomer emulsified solution;

(6) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), introducing nitrogen, cleaning for 5min, swelling for 20h at 25 ℃, wherein the heating rate is 1.5 ℃/min, then adding 0.5g of polyvinylpyrrolidone stabilizer and 0.025g of sodium nitrite aqueous phase polymerization inhibitor, heating the reaction temperature to 80 ℃, the heating rate is 1.5 ℃/min, reacting for 24h at the rotating speed of 200rpm, centrifuging for three times at the rotating speed of 3000rpm after the reaction is finished, and washing for multiple times by using deionized water to obtain the micron-sized AIE polystyrene fluorescent microsphere solution each time for 10 min.

The morphology of the AIE polystyrene fluorescent microspheres is observed through a scanning electron microscope, and the AIE polystyrene fluorescent microspheres can be observed to be spherical particles with the particle size of 10-12 microns. The fluorescence spectrum of the AIE polystyrene fluorescent microsphere shows that the emission wavelength of the AIE polystyrene fluorescent microsphere is 550nm, and the AIE polystyrene fluorescent microsphere is yellow fluorescence under the irradiation of an ultraviolet lamp. The fluorescent diatom screening test paper is used for researching the feasibility of the fluorescence-coupled diatom in drowning diagnosis, the distribution of AIE fluorescent diatom can be clearly and brightly observed under a fluorescent microscope, and the fluorescent signal value is 32509.

Example 3

(1) Weighing 5g of polyvinyl alcohol stabilizer, dissolving the polyvinyl alcohol stabilizer in a mixed solution of 40g of ethanol and 10g of water to obtain a stabilizer solution, adding the stabilizer solution into a 500mL four-neck flask, and stirring and preheating at the rotating speed of 150rpm at 70 ℃ for 20 min;

(2) weighing 5g of dibenzoyl peroxide initiator, adding the dibenzoyl peroxide initiator into 10g of styrene monomer, stirring and mixing at the rotating speed of 400rpm at 40 ℃, and uniformly mixing to obtain a monomer solution;

(3) adding the monomer solution into the stabilizer solution, introducing nitrogen to carry out sweeping for 15min, and stirring and reacting for 26h in an oil bath kettle at 90 ℃ at the rotating speed of 150 rpm; after the reaction is finished, centrifuging for three times at the rotating speed of 4000rpm, washing for 15min each time by deionized water for multiple times to obtain a polystyrene seed microsphere solution, determining the solid content and the conversion rate, and diluting the polystyrene seed microsphere solution to the concentration of 1%.

The morphology of the polystyrene seed microspheres is observed by a scanning electron microscope, the polystyrene seed microspheres are observed to be spherical particles with the particle size of 1.4 mu m, the conversion rate of the seed microspheres is measured by a gravimetric method, and the conversion rate of the seed microspheres is calculated to be 95%.

(4) Weighing 0.1g of dibutyl phthalate, adding the dibutyl phthalate into 50g of seed microsphere solution, placing the seed microsphere solution in an ice water bath, carrying out ultrasonic treatment for 5min at the power of 100W to obtain a swelling agent solution, adding the swelling agent solution into a four-neck flask, swelling for 24h at the temperature of 25 ℃, and obtaining an activated swelling solution at the temperature rise rate of 2 ℃/min and the rotation speed of 400 rpm;

(5) weighing 0.2g of sodium dodecyl sulfate, adding the sodium dodecyl sulfate into 100mL of ultrapure water to obtain an aqueous phase solution, weighing 0.5g of dibenzoyl peroxide initiator, 0.4g of methacrylic acid and 0.5g of AIE-2 molecules, adding the dibenzoyl peroxide initiator, the methacrylic acid and the AIE-2 molecules into 20g of styrene monomer, uniformly mixing, adding the aqueous phase solution, stirring at the rotating speed of 700rpm at 40 ℃ for 15min for pre-emulsification, placing a container filled with a coarse emulsion in an ice-water bath, and performing ultrasound at the power of 600W for 15min to obtain a monomer emulsified solution;

(6) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), introducing nitrogen, cleaning for 20min, swelling for 36h at 25 ℃, wherein the heating rate is 2 ℃/min, then adding 10g of polyethylene glycol stabilizer and 0.5g of sodium nitrite aqueous phase polymerization inhibitor, heating the reaction temperature to 90 ℃, reacting for 36h at the rotating speed of 400rpm, the heating rate is 2 ℃/min, centrifuging for three times at the rotating speed of 4000rpm after the reaction is finished, and washing for 20min each time by using deionized water for multiple times to obtain the micron-sized AIE polystyrene fluorescent microsphere solution.

The morphology of the AIE polystyrene fluorescent microspheres is observed through a scanning electron microscope, and the AIE polystyrene fluorescent microspheres are observed to be spherical particles with the particle size of 12-14 microns. The fluorescence spectrum of the AIE polystyrene fluorescent microsphere shows that the emission wavelength of the AIE polystyrene fluorescent microsphere is 625nm, and the AIE polystyrene fluorescent microsphere is red fluorescence under the irradiation of an ultraviolet lamp. The fluorescent diatom screening test is used for researching the feasibility of the fluorescence-coupled diatom in drowning diagnosis, the distribution of AIE fluorescent diatom can be clearly and brightly observed under a fluorescent microscope, and the fluorescent signal value is 28763.

Example 4

(1) Weighing 0.1g of polyvinyl alcohol stabilizer, dissolving in 50g of ethanol to obtain a stabilizer solution, adding the stabilizer solution into a 250mL four-neck flask, and stirring and preheating at 65 ℃ at the rotating speed of 70rpm for 20 min;

(2) weighing 1g of azodiisoheptanonitrile initiator, adding the azodiisoheptanonitrile initiator into 10g of methyl methacrylate monomer, stirring and mixing at the rotating speed of 400rpm at 40 ℃, and uniformly mixing to obtain monomer solution;

(3) adding the monomer solution into the stabilizer solution, introducing nitrogen to carry out sweeping for 15min, and stirring and reacting for 20h in an oil bath kettle at 65 ℃ at the rotating speed of 70 rpm; after the reaction is finished, centrifuging for three times at the rotating speed of 4000rpm, washing for 15min each time by deionized water for multiple times to obtain a polystyrene seed microsphere solution, determining the solid content and the conversion rate, and diluting the polymethyl methacrylate seed microsphere solution to the concentration of 1%.

The shape of the polymethyl methacrylate seed microspheres is observed by a scanning electron microscope, the polymethyl methacrylate seed microspheres are observed to be spherical particles, the particle size is 3.2 mu m, the conversion rate of the seed microspheres is measured by a gravimetric method, and the conversion rate of the seed microspheres is calculated to be 90%.

(4) Weighing 0.5g of dibutyl phthalate, adding the dibutyl phthalate into 15g of seed microsphere solution, placing the mixture into an ice water bath, carrying out ultrasonic treatment for 15min at the power of 500W to obtain swelling agent solution, adding the swelling agent solution into a four-neck flask, swelling for 15h at the temperature of 25 ℃, and obtaining activated swelling solution at the temperature rise rate of 2.5 ℃/min and the rotating speed of 300 rpm;

(5) weighing 0.2g of sodium dodecyl sulfate, adding the sodium dodecyl sulfate into 80mL of ultrapure water to obtain an aqueous phase solution, weighing 0.3g of azodiisoheptanonitrile initiator, 0.5g of methacrylic acid and 0.3g of AIE-2 molecules, adding the mixture into 10g of styrene monomer, uniformly mixing, adding the aqueous phase solution, stirring at the rotating speed of 700rpm at 40 ℃ for 15min for pre-emulsification, placing a container filled with a crude emulsion in an ice water bath, and performing ultrasound at the power of 500W for 15min to obtain a monomer emulsified solution;

(6) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), introducing nitrogen, cleaning for 15min, swelling for 30h at 25 ℃, wherein the heating rate is 2.5 ℃/min, then adding 3g of polyvinyl alcohol stabilizer and 0.1g of cuprous chloride aqueous phase polymerization inhibitor, raising the reaction temperature to 90 ℃, reacting for 30h at the rotating speed of 300rpm, wherein the heating rate is 2.5 ℃/min, centrifuging for three times at the rotating speed of 2000rpm after the reaction is finished, each time for 15min, and washing with deionized water for multiple times to obtain the micron-sized AIE polystyrene fluorescent microsphere solution.

The morphology of the AIE polystyrene fluorescent microspheres is observed through a scanning electron microscope, and the AIE polystyrene fluorescent microspheres can be observed to be spherical particles with the particle size of 8-10 microns. The fluorescence spectrum of the AIE polystyrene fluorescent microsphere shows that the emission wavelength of the AIE polystyrene fluorescent microsphere is 435nm, and the AIE polystyrene fluorescent microsphere is blue fluorescence under the irradiation of an ultraviolet lamp. The fluorescent diatom screening test is used for researching the feasibility of the fluorescence-coupled diatom in drowning diagnosis, the distribution of AIE fluorescent diatom can be clearly and brightly observed under a fluorescent microscope, and the fluorescent signal value is 35097.

Example 5

(1) Weighing 2g of polyvinyl alcohol stabilizer, dissolving in 50g of ethanol to obtain a stabilizer solution, adding the stabilizer solution into a 500mL four-neck flask, and stirring and preheating at 65 ℃ at a rotating speed of 50rpm for 20 min;

(2) weighing 2g of azobisisobutyronitrile initiator, adding the azobisisobutyronitrile initiator into 10g of styrene monomer, stirring and mixing at 40 ℃ at a rotating speed of 400rpm, and uniformly mixing to obtain a monomer solution;

(3) adding the monomer solution into the stabilizer solution, introducing nitrogen to carry out sweeping for 20min, and stirring and reacting for 15h in an oil bath kettle at the temperature of 60 ℃ at the rotating speed of 100 rpm; after the reaction is finished, centrifuging for three times at the rotating speed of 3000rpm, washing for 20min each time by deionized water for multiple times to obtain a polystyrene seed microsphere solution, determining the solid content and the conversion rate, and diluting the polystyrene seed microsphere solution to the concentration of 1%.

The morphology of the polystyrene seed microspheres is observed by a scanning electron microscope, the polystyrene seed microspheres are observed to be spherical particles with the particle size of 4.2 mu m, the conversion rate of the seed microspheres is measured by a gravimetric method, and the conversion rate of the seed microspheres is calculated to be 96%.

(4) Weighing 0.5g of dibutyl phthalate, adding the dibutyl phthalate into 20g of seed microsphere solution, placing the seed microsphere solution in an ice water bath, carrying out ultrasonic treatment for 20min at the power of 200W to obtain a swelling agent solution, adding the swelling agent solution into a four-neck flask, swelling for 3h at the temperature of 50 ℃, and obtaining an activated swelling solution at the temperature rise rate of 3 ℃/min and the rotation speed of 200 rpm;

(5) weighing 0.6g of sodium dodecyl sulfate, adding the sodium dodecyl sulfate into 120mL of ultrapure water to obtain an aqueous phase solution, weighing 0.4g of dibenzoyl peroxide initiator, 0.4g of methacrylic acid and 0.3g of AIE-2 molecule, adding the dibenzoyl peroxide initiator, the methacrylic acid and the AIE-2 molecule into 10g of styrene monomer, uniformly mixing, adding the aqueous phase solution, stirring at the rotating speed of 400rpm at 40 ℃ for 10min for pre-emulsification, placing a container filled with a coarse emulsion in an ice-water bath, and performing ultrasonic treatment at the power of 500W for 20min to obtain a monomer emulsified solution;

(6) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), introducing nitrogen, cleaning for 20min, swelling for 30h at 25 ℃, heating at a rate of 3 ℃/min, then adding 3g of polyvinyl alcohol stabilizer and 0.1g of cuprous chloride aqueous phase polymerization inhibitor, heating the reaction temperature to 85 ℃, heating at a rate of 3 ℃/min, reacting for 30h at a rotating speed of 300rpm, centrifuging for three times at a rotating speed of 2000rpm after the reaction is finished, each time for 15min, and washing with deionized water for multiple times to obtain the micron-sized AIE polystyrene fluorescent microsphere solution.

The morphology of the AIE polystyrene fluorescent microspheres is observed through a scanning electron microscope, and the AIE polystyrene fluorescent microspheres are observed to be spherical particles with the particle size of 13-15 microns. The fluorescence spectrum of the AIE polystyrene fluorescent microsphere shows that the emission wavelength of the AIE polystyrene fluorescent microsphere is 565nm, and the AIE polystyrene fluorescent microsphere is yellow fluorescence under the irradiation of an ultraviolet lamp. The fluorescent diatom screening test is used for researching the feasibility of the fluorescence-coupled diatom in drowning diagnosis, the distribution of AIE fluorescent diatom can be clearly and brightly observed under a fluorescent microscope, and the fluorescent signal value is 38672.

Example 6

(1) Weighing 0.1g of polyvinylpyrrolidone stabilizer, dissolving in 50g of ethanol to obtain a stabilizer solution, adding the stabilizer solution into a 500mL four-neck flask, and stirring and preheating at the rotating speed of 50rpm at 75 ℃ for 20 min;

(3) adding the monomer solution into the stabilizer solution, introducing nitrogen for sweeping for 5min, and stirring and reacting for 18h in a 75 ℃ oil bath kettle at the rotating speed of 80 rpm; after the reaction is finished, centrifuging for three times at 10000rpm for 20min each time, washing for multiple times by deionized water to obtain a polystyrene seed microsphere solution, determining the solid content and the conversion rate, and diluting the polystyrene seed microsphere solution to 1% concentration.

The morphology of the polystyrene seed microspheres is observed by a scanning electron microscope, the polystyrene seed microspheres are observed to be spherical particles with the particle size of 3.2 mu m, the conversion rate of the seed microspheres is measured by a gravimetric method, and the conversion rate of the seed microspheres is calculated to be 93%.

(4) Weighing 0.15g of dibutyl phthalate, adding the dibutyl phthalate into 25g of seed microsphere solution, placing the mixture into an ice water bath, carrying out ultrasonic treatment for 15min at the power of 300W to obtain swelling agent solution, adding the swelling agent solution into a four-neck flask, swelling for 3h at the temperature of 35 ℃, and obtaining activated swelling solution at the temperature rise rate of 1 ℃/min and the rotation speed of 200 rpm;

(5) weighing 0.8g of sodium dodecyl sulfate, adding the sodium dodecyl sulfate into 60mL of ultrapure water to obtain an aqueous phase solution, weighing 0.05g of azodiisoheptanonitrile initiator, 0.4g of methacrylic acid and 0.3g of AIE-2 molecules, adding the mixture into 20g of styrene monomer, uniformly mixing, adding the aqueous phase solution, stirring at the rotating speed of 400rpm at 40 ℃ for 10min for pre-emulsification, placing a container filled with a coarse emulsion in an ice water bath, and performing ultrasonic treatment at the power of 300W for 10min to obtain a monomer emulsified solution;

(6) and (3) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), introducing nitrogen, cleaning for 20min, swelling for 30h at 25 ℃, heating at a rate of 1 ℃/min, then adding 1.5g of polyvinylpyrrolidone stabilizer and 0.03g of cuprous chloride aqueous phase polymerization inhibitor, heating to 85 ℃, reacting for 20h at a rotation speed of 200rpm, heating at a rate of 1 ℃/min, centrifuging for three times at a rotation speed of 5000rpm after the reaction is finished, and washing for 5min each time by using deionized water for multiple times to obtain the AIE polystyrene fluorescent microsphere solution.

The morphology of the AIE polystyrene fluorescent microspheres is observed through a scanning electron microscope, and the AIE polystyrene fluorescent microspheres can be observed to be spherical particles with the particle size of 15-16 microns. The fluorescence spectrum of the AIE polystyrene fluorescent microsphere shows that the emission wavelength of the AIE polystyrene fluorescent microsphere is 660nm, and the AIE polystyrene fluorescent microsphere is red fluorescence under the irradiation of an ultraviolet lamp. The fluorescent coupled diatom fluorescence microscope is used for researching the feasibility of the fluorescence coupled diatom in drowning diagnosis, the distribution of AIE fluorescent diatom can be clearly and brightly observed under a fluorescent microscope, and the fluorescence signal value is 28732.

Example 7

(1) Weighing 4g of polyethylene glycol stabilizer, dissolving the polyethylene glycol stabilizer in a mixed solution of 40g of ethanol and 10g of water to obtain a stabilizer solution, adding the stabilizer solution into a 500mL four-neck flask, and stirring and preheating at 600 ℃ at the rotating speed of 200rpm for 20 min;

(2) weighing 4g of azobisisobutyronitrile initiator, adding the azobisisobutyronitrile initiator into 15g of methyl methacrylate monomer, stirring and mixing at 40 ℃ at a rotating speed of 400rpm, and uniformly mixing to obtain a monomer solution;

(3) adding the monomer solution into the stabilizer solution, introducing nitrogen to sweep for 30min, and stirring and reacting for 18h in an oil bath kettle at the temperature of 60 ℃ at the rotating speed of 80 rpm; after the reaction is finished, centrifuging for three times at the rotating speed of 4000rpm, washing for 10min each time by deionized water for multiple times to obtain a polystyrene seed microsphere solution, determining the solid content and the conversion rate, and diluting the polymethyl methacrylate seed microsphere solution to the concentration of 1%.

The shape of the polymethyl methacrylate seed microspheres is observed by a scanning electron microscope, the polymethyl methacrylate seed microspheres are observed to be spherical particles, the particle size is 4.2 mu m, the conversion rate of the seed microspheres is measured by a gravimetric method, and the conversion rate of the seed microspheres is calculated to be 94%.

(4) Weighing 0.35g of dibutyl phthalate, adding the dibutyl phthalate into 30g of seed microsphere solution, placing the mixture into an ice water bath, carrying out ultrasonic treatment for 5min at 650W to obtain swelling agent solution, adding the swelling agent solution into a four-neck flask, swelling for 4h at 40 ℃, heating at a rate of 1.5 ℃/min, rotating at a speed of 200rpm to obtain activated swelling solution;

(5) weighing 0.8g of sodium dodecyl sulfate, adding the sodium dodecyl sulfate into 150mL of ultrapure water to obtain an aqueous phase solution, weighing 0.4g of azodiisoheptanonitrile initiator, 0.5g of methacrylic acid and 0.5g of AIE-2 molecules, adding the mixture into 20g of styrene monomer, uniformly mixing, adding the aqueous phase solution, stirring at the rotating speed of 400rpm at 40 ℃ for 10min for pre-emulsification, placing a container filled with a coarse emulsion in an ice water bath, and performing ultrasonic treatment at the power of 650W for 5min to obtain a monomer emulsified solution;

(6) adding the monomer emulsified solution obtained in the step (5) into the activated swelling solution obtained in the step (4), introducing nitrogen, cleaning for 20min, swelling for 30h at 25 ℃, heating at a rate of 1.5 ℃/min, then adding 8g of polyethylene glycol stabilizer and 0.3g of sodium nitrite aqueous phase polymerization inhibitor, heating the reaction temperature to 90 ℃, heating at a rate of 1.5 ℃/min, reacting for 20h at a rotating speed of 200rpm, centrifuging for three times at a rotating speed of 2000rpm after the reaction is finished, each time for 30min, and washing with deionized water for multiple times to obtain the micron-sized AIE polystyrene fluorescent microsphere solution.

The morphology of the AIE polystyrene fluorescent microspheres is observed through a scanning electron microscope, and the AIE polystyrene fluorescent microspheres can be observed to be spherical particles with the particle size of 10-12 microns. The fluorescence spectrum of the AIE polystyrene fluorescent microsphere shows that the emission wavelength is 515nm, and the microsphere is green fluorescence under the irradiation of an ultraviolet lamp. The fluorescent diatom screening test paper is used for researching the feasibility of the fluorescence-coupled diatom in drowning diagnosis, the distribution of AIE fluorescent diatom can be clearly and brightly observed under a fluorescent microscope, and the fluorescence signal value is 19765.

The above-described embodiments of the invention are intended to be illustrative of the invention and are not to be construed as limiting the invention, and any variations that fall within the meaning and scope of the invention equivalent to the claims are intended to be embraced therein.

Claims

1. A preparation method of micron-sized aggregation-induced emission polymer microspheres is characterized by comprising the following steps:

(1) dissolving a stabilizer in a solvent to obtain a stabilizer solution;

2. The method for preparing micro-sized aggregation-induced emission polymer microspheres according to claim 1,

preheating the stabilizer solution in the step (1) at 50-80 ℃ for 10-40 min;

the mass of the stabilizer in the step (1) is 0.1-10% of that of the solvent;

the stabilizing agent in the step (1) is selected from one of the following: polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, dextrin, polyethylene glycol;

the solvent in the step (1) is selected from one of methanol, ethanol, propanol, butanol, pentanol or a mixed solution of ethanol and water.

3. The method for preparing micro-sized aggregation-induced emission polymer microspheres according to claim 1,

the hydrophobic monomer in the step (2) accounts for 5-40% of the mass of the solvent in the step (1), and the initiator accounts for 0.1-10% of the mass of the solvent in the step (1);

the hydrophobic monomer in the step (2) is selected from one of the following: styrene, acrylic acid, methyl acrylate, methyl methacrylate, methacrylic acid;

the initiator in the step (2) is selected from one of the following: azobisisobutyronitrile, dibenzoyl peroxide, azobisisovaleronitrile, cumene hydroperoxide, azobisisoheptonitrile, tert-butyl peroxypivalate, diisopropyl peroxydicarbonate, and dilauroyl peroxide.

4. The method for preparing micro-sized aggregation-induced emission polymer microspheres according to claim 1,

the heating and stirring temperature in the step (3) is 40-90 ℃, and the time is 16-36 h;

the speed of the centrifugal purification in the step (3) is 1000 rpm-5000 rpm, and the time of the centrifugal purification is 3 min-40 min;

the mass fraction of the seed microsphere solution in the step (3) is 0.5-1.5%.

5. The method for preparing micro-sized aggregation-induced emission polymer microspheres according to claim 1,

the swelling agent accounts for 0.1-5% of the seed microsphere solution in mass in the step (4);

the power of the ultrasonic treatment in the step (4) is 25W-950W, the time is 0.5 min-60 min, and the temperature is 0 ℃ to 5 ℃;

the temperature of the swelling treatment in the step (4) is 20-60 ℃, and the time is 6-24 h;

the swelling agent in the step (4) is selected from one of the following: dibutyl phthalate, trichloromethane, dichloromethane, toluene, tetrahydrofuran, acetone and cyclohexane.

6. The method for preparing micro-sized aggregation-induced emission polymer microspheres according to claim 1,

the power of the ultrasonic treatment in the step (5) is 25W-950W, and the time is 0.5 min-60 min;

in the step (5), the mass of the surfactant is 0.1-10% of the mass of water, the mass of the functional monomer is 0.1-50% of the mass of the hydrophobic monomer, the mass of the AIE molecule is 0.5-50% of the mass of the hydrophobic monomer, the mass of the initiator is 0.1-10% of the mass of the hydrophobic monomer, and the mass of the hydrophobic monomer is 2-30% of the mass of water.

7. The method for preparing micro-sized aggregation-induced emission polymer microspheres according to claim 1,

the surfactant in the step (5) is selected from one of the following: sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, dodecyl amino propionic acid, octadecyl dihydroxyethyl amine oxide, and tween-20;

the hydrophobic monomer in the step (5) is selected from one of the following: styrene, methyl acrylate, methyl methacrylate;

the functional monomer in the step (5) is selected from one of the following: acrylic acid, methacrylic acid, hydroxyethyl methacrylate, itaconic acid;

the AIE molecule of step (5) is at least one selected from the following AIE-1 to AIE-18 molecules:

the initiator is selected from one of the following: azobisisobutyronitrile, dibenzoyl peroxide, azobisisovaleronitrile, cumene hydroperoxide, azobisisoheptonitrile, tert-butyl peroxypivalate, diisopropyl peroxydicarbonate, and dilauroyl peroxide.

8. The method for preparing micro-sized aggregation-induced emission polymer microspheres according to claim 1,

the swelling temperature in the step (6) is 20-60 ℃, and the swelling time is 3-10 h;

the temperature rise rate of the swelling in the step (6) is 0.5-5 ℃/min;

the reaction temperature in the step (6) is 40-100 ℃, and the reaction time is 10-36 h;

the stabilizer in the step (6) is selected from one of the following: polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol;

the aqueous phase polymerization inhibitor in the step (6) is selected from one of the following: cuprous chloride and sodium nitrite.

9. Micron-sized aggregation-inducing luminescent polymer microspheres prepared by the preparation method of any one of claims 1 to 8.

10. Use of the micro-sized aggregation-inducing luminescent polymer microspheres of claim 9 for the preparation of conjugated biomolecules for fluorescence tracking.